We have recently identified the repressive histone variant macroH2A1.2 as a critical modulator of BRCA1-dependent genome maintenance during DSB repair via homologous recombination (HR). Given that both HR and BRCA1 function are required for the efficient resolution of stalled and/or collapsed replication forks, we next asked if macroH2A1.2 may act to modulate this process, invoking chromatin as a novel paradigm for the manipulation of the cellular response to replication stress. Using chromatin immunoprecipitation combined with deep sequencing (ChIP-Seq) in K562 erythroleukemia cells, in which both fragile sites an DNA replication patterns have been extensively characterized (79-81), we were able to show that macroH2A1.2 localizes to sites of replication stress-induced DNA damage. Notably, macroH2A1.2 peak coverage was most prominent at common fragile sites and was further positively correlated with CFS susceptibility to DNA breaks. Consistent with an active role during replication stress, we observed a fragile-site specific increase in macroH2A1.2 beyond its basal level of enrichment, which was dependent on DNA damage signaling via ATR/ATM. Other H2A variants, including macroH2A1.1 and H2A.Z, remained unaltered, indicating that macroH2A.1.2 plays a unique role in the cellular response to replication stress. Demonstrating functional significance of this observation, we identified a protective role for macroH2A1.2 during replication stress, preventing excessive DNA damage accumulation at fragile DNA. Consistent with our finding that macroH2A1.2 promotes BRCA1-dependent genome maintenance at DSBs, we observed a significant reduction in BRCA1 localization at nascent replication forks using iPOND. Moreover, and in agreement with our ChIP results at fragile sites, replication stress resulted in a pronounced increase in gamma-H2AX at stalled forks in macroH2A1.2-depleted cells. Together, these findings demonstrate macroH2A1.2-dependent recruitment and/or stabilization of BRCA1 at replication forks, which is in turn protects from replication stress. While beneficial for genome maintenance in the short term, replication stress-induced chromatin reorganization may be a driver of progressive epigenetic dysfunction, particularly in the context of replicative age. To test this possibility, we used human primary fibroblasts, which can be cultured for a finite number of population doublings (PDs) and exhibit profound, yet poorly understood chromatin changes in late passage cells. We observed a robust, replication-dependent increase in macroH2A1.2 at seven of eight tested CFSs, that correlated with both age and replication stress-associated gene deregulation. Importantly, the same fragile sites were also subject to macroH2A1.2 and gamma-H2AX accumulation in response to replication stress. Together this work establishes macroH2A1.2 as a bona fide epigenetic modulator of replication stress with implications for age-associated epigenetic decline. Notably, replication stress and the resulting DNA damage response (DDR) are important drivers of cellular senescence, thus presenting an important barrier to malignant transformation. Consistent with a role for macroH2A1.2 in this process, our preliminary data indicate that loss of macroH2A1.2 can cause a near complete cell cycle arrest in primary fibroblasts, followed by hallmarks of cellular senescence. The molecular basis for this finding is under investigation. Together, this work is expected to have significant implications for our understanding of both genome maintenance and malignant transformation in dividing cells. As an orthogonal approach to study the impact of DNA damage on epigenetic integrity, we have generated a mouse model that allows for inducible, tissue-specific DSB formation at 140 defined genomic loci. While DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways, studying theconsequences of DSBs in higher organisms has been hindered by a scarcity of tools for controlled DSB induction. Using this novel mouse model, we found that DSBs promote a DNA damage signaling-dependent decrease in gene expression in primary cells, which occurs specifically at break-bearing genes and is reversed upon repair of the lesion. Importantly, we demonstrate that the restoration of gene expression can occur independently of cell cycle progression, underlining its relevance for normal tissue maintenance and extending previous findings based on the analysis of tumor cell lines in vivo. Consistent with the reversal of gene expression changes in vitro, we observed no evidence for persistent transcriptional repression in response to a three-day course of continuous DSB formation and repair in mouse lymphocytes in vivo. Together, our findings reveal an unexpected capacity of primary cells to maintain transcriptome integrity in response to short term DSB exposure, pointing to a limited role for DNA damage as a mediator of cell-autonomous epigenetic dysfunction. This work was recently published in Nucleic Acids Research.